1. Field of the Invention
The present invention relates to the activation and stabilization of reagents used in enzymatic assays. In particular, the invention relates to the use of propylene glycol, at suitable pH levels, to increase the specific activity of the phosphoenolpyruvate carboxylase (PEPC) enzyme reagent used in carbon dioxide assays.
2. Description of Related Art
The total carbon dioxide content of serum consists primarily of bicarbonate ions (HCO.sub.3.sup.-) which account for 90 to 95 percent of the total carbon dioxide (CO.sub.2) content, and the remaining serum CO.sub.2 is present in the physically dissolved state. Bicarbonate has a normal serum level range of 23 to 32 meq/L and is the second largest fraction of anions present in serum. Clinically, an alteration of the serum bicarbonate level is reflective of an acid-base imbalance. An analysis of the bicarbonate concentration, together with an evaluation of electrolytes and other blood gases, will give an overall picture of the acid-base imbalance.
A typical carbon dioxide assay utilizes PEPC and an enzymatic reaction to determine the total carbon dioxide concentration. ("Reagents for Enzymatic Analysis," in Methods of Enzymatic Analysis, 3d ed. Bergmeyer [Weinheim: Verlag Chemie, 1983], 275-76.) The assay can be depicted by the following reactions: ##STR1## wherein P.sub.i is inorganic phosphorus, NADH is the reduced form of nicotinamide adenine dinucleotide, MDH is malate dehydrogenase and NAD.sup.+ is the oxidized form of nicotinamide adenine dinucleotide. The two step reaction can be monitored by measuring the disappearance of NADH at the bichromatic wavelength of 340/380 nanometers (nm), i.e., the decrease in NADH concentration is proportional to the amount of carbon dioxide in the sample. Alternatively, the first enzyme reaction can be monitored by measuring either the quantity of oxaloacetate produced or the rate at which the oxaloacetate is produced.
Reagent stability is a critical problem with assay reagents utilized to determine total carbon dioxide concentration. Conventional PEPC enzyme reagent formulations are stable for only about four to eight hours. Carbon dioxide in solution is in a state of equilibrium between dissolved carbon dioxide and carbonic acid which dissociates to form H.sup.+ and HCO.sub.3.sup.- in accordance with the following equation: EQU CO.sub.2 (dissolved)+H.sub.2 O.revreaction.H.sub.2 CO.sub.3 .revreaction.H.sup.+ +HCO.sub.3.sup.-
("Carbon Dioxide and Bicarbonate," in Manometric & Biochemical Techniques, ed. Umbreit [Minneapolis: Burgess Publishing Co., 1972], 20. The reagents are destabilized by the absorption of carbon dioxide from the atmosphere; the CO.sub.2 enters the reaction sequence and consumes the phosphoenolpyruvate and NADH present in the reagent. Conventional assays are based upon the principle that a pH of about 7.5 to about 10.5 is necessary for the conversion of both dissolved CO.sub.2 and carbonic acid to the bicarbonate ion, as disclosed by Adams in U.S. Pat. No. 3,974,037. It is now believed that the actual substrate of the PEPC enzyme is CO.sub.2. (Waygood et al., Can. J. Bot. (1969) 47: 1455-58.)
There is a need, therefore, for a PEPC enzyme reagent which is stable in the atmosphere under normal conditions of use and which has a pH level suitable for use in an assay designed to measure bicarbonate as a function of CO.sub.2 concentration.